Die Interaktion des Sauren Sphingomyelinase/Ceramid-Systems und TRPC6 Ionen-Kanälen in neuronalen Zellen

Recent clinical and murine studies indicated a critical role of the acid sphingomyelinase (ASM)/ceramide system in the pathogenesis of major depression (MD) (Kornhuber et al. 2005; Gulbins et al. 2013; Rhein et al. 2017). It was shown that the pharmacological antidepressive effects of several commonly used antidepressants, as fluoxetine, depend on the functional inhibition of ASM (Kornhuber et al. 2011; Gulbins et al. 2013). Further, the amount of ceramide molecules in the plasma membrane regulates the clustering of membrane proteins that influences signalling via microdomain and lipid raft organization (Zhang et al. 2009; Grassmè, Riethmüller, and Gulbins 2007; Stancevic and Kolesnick 2010). TRPC6 ion channels are located in the plasma membrane and are crucially involved in synaptic plasticity (Li et al. 2005; Tai et al. 2008; Leuner et al. 2013). Synaptic plasticity has been shown to be compromised in MD (Duman et al. 2016). Therefore the question was, if a dysregulation of the ASM/ceramide system might impact the TRPC6 functioning. Since antidepressants induce an inhibition of ASM activity, I aimed at investigating their consequences for TRPC6 function. I could show that some antidepressants and the direct ASM inhibitor ARC39 inhibited ASM activity, but the experimental parameters differed regarding to cell types. The reduction in ASM activity also affected TRPC6 biology: antidepressant treatment induced TRPC6 protein expression in PC12 cells and rat cortical neurons, and the genetic ablation of ASM in a mouse model affected TRPC6 RNA levels in a sex-specific pattern. Inhibition of ASM activity was found to impair the TRPC6-mediated Ca2+ influx in PC12 cells and in ASM-deficient cortical mouse neurons. Moreover, the TRPC6 downstream effector - activated transcriptional factor phospho-CREB - was reduced in ASM-deficient and ASM-inhibited neurons. A lipidomic analysis of PC12 cells, which were inhibited in ASM activity, revealed a decrease in ceramide/sphingomyelin ratio in particular for very-long chain sphingolipid species. This affected TRPC6 localisation to lipid rafts and non-raft regions at the plasma membrane. Deductively, disturbed localisation of TRPC6 at the plasma membrane, due to ASM inhibition and subsequent decrease of ceramide levels, could serve as a mechanistical explanation for observed impaired TRPC6-mediated Ca2+ influx and downstream signalling. In summary, the better understanding of the interaction of the ASM/ceramide system and TRPC6 will help to draw conclusions for the pathogenesis of this disease. My primary cell data together with other previous findings in PC12 (Ye 2015) lead to the hypothesis that a sensitive ceramide balance is necessary for proper functioning of TRPC6 in primary cells. It can be speculated that an increase in ASM activity, that was correlated with MD, also results in the dysfunction of TRPC6 and could be rescued by antidepressant mediated ASM inhibition via restoration of physiological sphingolipid balance. Further experiments with ASM-transgenic animals will help to elucidate these questions in the future.Aktuelle klinische und tierexperimentelle Studien zeigen, dass das saure Sphingomyelinase (ASM)/Ceramid-System eine bedeutende Rolle in der Pathogenese der Majoren Depression (MD) spielt (Kornhuber et al. 2005; Gulbins et al. 2013; Rhein et al. 2017) und der antidepressive Effekt verschiedener allgemein gebräuchlicher Antidepressiva von der Inhibierung der ASM abhängt (Kornhuber et al. 2011; Gulbins et al. 2013). Das Ceramid-System hat zudem eine wichtige Bedeutung für die Zusammensetzung der Plasmamembran. Ein erhöhtes Vorkommen von Ceramid-Molekülen führt zur Bildung von „Lipid Rafts“ und geht mit einer Veränderung der Membranstruktur und der Signaltransduktion einher (Zhang et al. 2009; Grassmè, Riethmüller, and Gulbins 2007; Stancevic and Kolesnick 2010). Die Signaltransduktion der in der Plasmamembran lokalisierten TRPC6 Ionen Kanälen ist u.a. für synaptische Plastizität von Bedeutung (Li et al. 2005; Tai et al. 2008; Leuner et al. 2013), welche bei MD verändert ist (Duman et al. 2016). Daher stellte sich die Frage, ob eine bei MD vorliegende Dysregulation des ASM/Ceramid-Systems die TRPC6 Funktion beeinflusst. Da Antidepressiva die ASM-Aktivität inhibieren, wurden die Auswirkungen der ASM-Hemmung auf die TRPC6 Funktion untersucht. Es konnte sowohl in einer neuronalen Zelllinie als auch in primären Neuronen gezeigt werden, dass einige Antidepressiva und der direkte ASM-Hemmer ARC39 die ASM-Aktivität inhibieren, wobei eine Zell-spezifische Signatur vorlag. Die Reduktion der ASM-Aktivität wirkte sich zudem auf die Expression von TRPC6 aus: Antidepressive Behandlung oder genetische ASM-Defizienz veränderte die Expression von TRPC6 auf RNA oder Protein-Ebene. Durch die Hemmung der ASM-Aktivität wurde der durch TRPC6 vermittelte Calciumeinstrom in PC12-Zellen und in ASM defizienten kortikalen Mausneuronen inhibiert. Außerdem wurde durch die ASM-Inhibierung die Phosphorylierung des Transkriptionsfaktors CREB, der durch TRPC6 aktiviert wird, in Neuronen reduziert. Eine Analyse des Lipidoms von mit Antidepressiva behandelten Zellen, ließ die Abnahme des Ceramid/Sphingomyelin-Verhältnisses für „very-long chain“ Sphingolipidspezies erkennen, welches die Lokalisation von TRPC6 in „Lipid Raft“- und „Nicht-Raft“-Regionen in der Plasmamembran beeinflusste. Schlussfolgernd kann die ASM Hemmung, und die damit einhergehende verringerte Ceramid-Konzentration, die Lokalisation von TRPC6 in der Plasmamembran stören, was als mechanistische Erklärung für die Veränderungen im TRPC6-vermittelten Calciumeinstrom und der Signaltransduktion dienen kann. Zusammenfassend kann das bessere Verständnis der Interaktion zwischen dem ASM/Ceramid-System und TRPC6 Ableitungen für die Pathogenese der MD zulassen. Meine in primären Neuronen gewonnenen Daten, in Kombination mit anderen vorrausgegangenen Daten in PC12-Zellen (Ye 2015), lassen die Hypothese zu, dass ein ausbalanciertes Gleichgewicht von Ceramid nötig ist, um die physiologische TRPC6 Funktion in primären Zellen zu bewahren. Ein Anstieg der ASM Aktivität, wie er bei einer MD vorliegt, könnte somit ebenfalls in einer Dysfunktion von TRPC6 resultieren. Diese könnte jedoch durch eine Antidepressiva-vermittelte ASM Hemmung ausgeglichen werden, indem die physiologische Sphingolipid-Balance wiederhergestellt wird. Weitere Experimente mit ASM-transgenen Tieren werden helfen, diese Fragen zu beantworten

Acid Sphingomyelinase Impacts Canonical Transient Receptor Potential Channels 6 (TRPC6) Activity in Primary Neuronal Systems

The acid sphingomyelinase (ASM)/ceramide system exhibits a crucial role in the pathology of major depressive disorder (MDD). ASM hydrolyzes the abundant membrane lipid sphingomyelin to ceramide that regulates the clustering of membrane proteins via microdomain and lipid raft organization. Several commonly used antidepressants, such as fluoxetine, rely on the functional inhibition of ASM in terms of their antidepressive pharmacological effects. Transient receptor potential canonical 6 (TRPC6) ion channels are located in the plasma membrane of neurons and serve as receptors for hyperforin, a phytochemical constituent of the antidepressive herbal remedy St. John’s wort. TRPC6 channels are involved in the regulation of neuronal plasticity, which likely contributes to their antidepressant effect. In this work, we investigated the impact of reduced ASM activity on the TRPC6 function in neurons. A lipidomic analysis of cortical brain tissue of ASM deficient mice revealed a decrease in ceramide/sphingomyelin molar ratio and an increase in sphingosine. In neurons with ASM deletion, hyperforin-mediated Ca2+-influx via TRPC6 was decreased. Consequently, downstream activation of nuclear phospho-cAMP response element-binding protein (pCREB) was changed, a transcriptional factor involved in neuronal plasticity. Our study underlines the importance of balanced ASM activity, as well as sphingolipidome composition for optimal TRPC6 function. A better understanding of the interaction of the ASM/ceramide and TRPC6 systems could help to draw conclusions about the pathology of MDD

Aβ1-16 controls synaptic vesicle pools at excitatory synapses via cholinergic modulation of synapsin phosphorylation

Amyloid beta (Aβ) is linked to the pathology of Alzheimer’s disease (AD). At physiological concentrations, Aβ was proposed to enhance neuroplasticity and memory formation by increasing the neurotransmitter release from presynapse. However, the exact mechanisms underlying this presynaptic effect as well as specific contribution of endogenously occurring Aβ isoforms remain unclear. Here, we demonstrate that Aβ1-42 and Aβ1-16, but not Aβ17-42, increased size of the recycling pool of synaptic vesicles (SV). This presynaptic effect was driven by enhancement of endogenous cholinergic signalling via α7 nicotinic acetylcholine receptors, which led to activation of calcineurin, dephosphorylation of synapsin 1 and consequently resulted in reorganization of functional pools of SV increasing their availability for sustained neurotransmission. Our results identify synapsin 1 as a molecular target of Aβ and reveal an effect of physiological concentrations of Aβ on cholinergic modulation of glutamatergic neurotransmission. These findings provide new mechanistic insights in cholinergic dysfunction observed in AD

Überprüfung der lichten Weiten von Gitterstäben in der Pferdehaltung

An Gitterstäben, die in der Pferdehaltung an verschiedenen Stellen verwendet werden, können sich Pferde verletzen, wenn sie ihre Köpfe oder ihre Hufe hindurchstecken und nicht zurückziehen können. Um das Verletzungsrisiko zu reduzieren sind lichte Weiten und Materialstärken von Gitterstäben so zu wählen, dass Pferdeköpfe und -hufe entweder nicht zwischen den Freiräumen hindurchpassen oder aber gefahrlos wieder zurückgezogen werden können. Die bisherige Forschung liefert jedoch keine belastbaren Aussagen zu Stababständen (senkrecht und waagerecht), die für Pferde ungefährlich sind. Die in der Praxis verwendeten und in der Literatur empfohlenen Gitterstababstände beruhen auf Erfahrungswerten und technischen Materialeigenschaften. In der vorliegenden Untersuchung wurden Pferdeköpfe und -hufe von insgesamt 480 Pferden (233 Stuten, 204 Wallache und 43 Hengste) von 23 verschiedenen Rassen vermessen, um auf Grundlage der Anatomie der Pferde Aussagen über die Eignung von marktüblichen Stababständen in der Praxis treffen zu können. Es stellte sich heraus, dass bei senkrechten Gitterstäben eine lichte Weite von nicht mehr als 5 cm für alle Pferde ab einem Stockmaß von 110 cm und einem Alter von zwei Jahren als sicher bezeichnet werden kann. Bei waagerechten Gitterstäben erwies sich eine lichte Weite von genau 17 cm als sicher. Dies gilt für alle Pferde ab einem Alter von zwei Jahren oder ab einem Stockmaß von 148 cm. Kritisch sind die lichten Weiten von Panels zu beurteilen. Hier zeigte sich, dass die handelsüblichen Abstände der Gitterstäbe für die meisten Pferde eine erhebliche Gefahr darstellen. Wenn die Pferde beispielsweise versuchen außerhalb der Panels zu fressen und dabei ihren Kopf durch die Gitterstäbe stecken, kann es leicht passieren, dass sie sich mit dem Kopf zwischen den Gitterstäben verklemmen

Aβ1-16 controls synaptic vesicle pools at excitatory synapses via cholinergic modulation of synapsin phosphorylation

Amyloid beta (Aβ) is linked to the pathology of Alzheimer's disease (AD). At physiological concentrations, Aβ was proposed to enhance neuroplasticity and memory formation by increasing the neurotransmitter release from presynapse. However, the exact mechanisms underlying this presynaptic effect as well as specific contribution of endogenously occurring Aβ isoforms remain unclear. Here, we demonstrate that Aβ1-42 and Aβ1-16, but not Aβ17-42, increased size of the recycling pool of synaptic vesicles (SV). This presynaptic effect was driven by enhancement of endogenous cholinergic signalling via α7 nicotinic acetylcholine receptors, which led to activation of calcineurin, dephosphorylation of synapsin 1 and consequently resulted in reorganization of functional pools of SV increasing their availability for sustained neurotransmission. Our results identify synapsin 1 as a molecular target of Aβ and reveal an effect of physiological concentrations of Aβ on cholinergic modulation of glutamatergic neurotransmission. These findings provide new mechanistic insights in cholinergic dysfunction observed in AD

Acid sphingomyelinase – a regulator of canonical transient receptor potential channel 6 (TRPC6) activity

Recent investigations propose the acid sphingomyelinase (ASM)/ceramide system as a novel target for antidepressant action. ASM catalyzes the breakdown of the abundant membrane lipid sphingomyelin to the lipid messenger ceramide. This ASM‐induced lipid modification induces a local shift in membrane properties, which influences receptor clustering and downstream signaling. Canonical transient receptor potential channels 6 (TRPC6) are non‐selective cation channels located in the cell membrane that play an important role in dendritic growth, synaptic plasticity and cognition in the brain. They can be activated by hyperforin, an ingredient of the herbal remedy St. John’s wort for treatment of depression disorders. Because of their role in the context of major depression, we investigated the crosstalk between the ASM/ceramide system and TRPC6 ion channels in a pheochromocytoma cell line 12 neuronal cell model (PC12 rat pheochromocytoma cell line). Ca2+ imaging experiments indicated that hyperforin‐induced Ca2+ influx through TRPC6 channels is modulated by ASM activity. While antidepressants, known as functional inhibitors of ASM activity, reduced TRPC6‐mediated Ca2+ influx, extracellular application of bacterial sphingomyelinase rebalanced TRPC6 activity in a concentration‐related way. This effect was confirmed in whole‐cell patch clamp electrophysiology recordings. Lipidomic analyses revealed a decrease in very long chain ceramide/sphingomyelin molar ratio after ASM inhibition, which was connected with changes in the abundance of TRPC6 channels in flotillin‐1–positive lipid rafts as visualized by western blotting. Our data provide evidence that the ASM/ceramide system regulates TRPC6 channels likely by controlling their recruitment to specific lipid subdomains and thereby fine‐tuning their physical properties

mRNA Expression of SMPD1 Encoding Acid Sphingomyelinase Decreases upon Antidepressant Treatment

Major depressive disorder (MDD) is a severe psychiatric condition with key symptoms of low mood and lack of motivation, joy, and pleasure. Recently, the acid sphingomyelinase (ASM)/ceramide system has been implicated in the pathogenesis of MDD. ASM is a lysosomal glycoprotein that catalyzes the hydrolysis of sphingomyelin, an abundant component of membranes, into the bioactive sphingolipid ceramide, which impacts signaling pathways. ASM activity is inhibited by several common antidepressant drugs. Human and murine studies have confirmed that increased ASM activity and ceramide levels are correlated with MDD. To define a molecular marker for treatment monitoring, we investigated the mRNA expression of SMPD1, which encodes ASM, in primary cell culture models, a mouse study, and a human study with untreated MDD patients before and after antidepressive treatment. Our cell culture study showed that a common antidepressant inhibited ASM activity at the enzymatic level and also at the transcriptional level. In a genetically modified mouse line with depressive-like behavior, Smpd1 mRNA expression in dorsal hippocampal tissue was significantly decreased after treatment with a common antidepressant. The large human study showed that SMPD1 mRNA expression in untreated MDD patients decreased significantly after antidepressive treatment. This translational study shows that SMPD1 mRNA expression could serve as a molecular marker for treatment and adherence monitoring of MDD